Human immunodeficiency virus type-1 integrase containing a glycine to serine mutation at position 140 is attenuated for catalysis and resistant to integrase inhibitors

Abstract

L-chicoric acid (L-CA) is a potent inhibitor of HIV integrase (IN) in vitro. In this report, the effects of a glycine to serine mutation at position 140 (G140S) on HIV IN and its effects on IN inhibitor resistance are described. HIV containing the G140S mutation showed a delay in replication. Using real-time polymerase chain reaction, the delay was secondary to a failure in integration. The mutant protein (IN(G140S)) was attenuated approximately four-fold for catalysis under equilibrium conditions compared to wild-type IN (IN(WT)) and attenuated five-fold in steady-state kinetic analysis of disintegration. Fifty percent inhibitory concentration assays were performed with IN inhibitors against both IN proteins in disintegration and strand transfer reactions. IN(G140S) was resistant to both L-CA and L-731,988, a diketoacid. HIV containing the mutation was resistant to both inhibitors as well. The G140S mutation attenuates IN activity and confers resistance to IN inhibitors, suggesting that diketoacids and L-CA interact with a similar binding site on HIV IN.

Human Immunodeficiency Virus Type 1 (HIV-1) Integrase: Resistance to Diketo Acid Integrase Inhibitors Impairs HIV-1 Replication and Integration and Confers Cross-Resistance to l -Chicoric Acid

“…IN genes generated by PCR site-directed mutagenesis were amplified using Pfu polymerase (Stratagene) and primers that allow the IN gene to be inserted into HIV NL4-3 containing XbaI and SacII engineered restriction enzyme sites (37). This results in replacement of the HIV NL4-3 IN gene by mutant IN (35,37,47,48).…”

“…The samples were incubated at 60°C for 1 h and heat inactivated at 95°C for 15 min. DNA from each sample was amplified by SYBR Green-I PCR as previously described (35,49,61). Primers used were AA55 and M667, which amplify minus-strand strongstop DNA (69); M661 and M667, which amplify products of complete cDNA synthesis (69); and MH535 and MH536, which amplify two-LTR-circle DNA, the products of failed integration (8).…”

“…A glycine-140-to-serine point mutation within HIV IN confers complete resistance to L-CA in tissue culture (37), and recombinant IN containing this mutation is resistant in vitro (35). This point mutation also confers cross-resistance to L-731,988, both in tissue culture and in vitro (35).…”

“…A glycine-140-to-serine point mutation within HIV IN confers complete resistance to L-CA in tissue culture (37), and recombinant IN containing this mutation is resistant in vitro (35). This point mutation also confers cross-resistance to L-731,988, both in tissue culture and in vitro (35). Computer modeling of L-CA into the crystal structure of the catalytic core of IN has identified a putative binding pocket involving amino acids D64, C65, T66, H67, E92, D116, N117, Q148, E152, K156, and K159 (53,56).…”

“…Furthermore, the site of any replication defects was identified using SYBR Green-I real-time PCR, which is a highly quantitative measure of HIV replication and can identify the site of a replication defect (35,61). IN proteins containing each mutation were also evaluated for enzymatic activity in the disintegration and 3Ј-end-processingstrand transfer reactions.…”